Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli

Abstract

During the diauxic shift, Escherichia coli exhausts glucose and adjusts its expression pattern to grow on a secondary carbon source. Transcriptional profiling studies of glucose–lactose diauxic transitions reveal a key role for ppGpp. The amount of ppGpp depends on RelA synthetase and the balance between a strong SpoT hydrolase and its weak synthetase. In this study, mutants are used to search for synthetase or hydrolase specific regulation. Diauxic shifts experiments were performed with strains containing SpoT hydrolase and either RelA or SpoT synthetase as the sole source of ppGpp. Here, the length of the diauxic lag times is determined by the presence of ppGpp, showing contributions of both ppGpp synthetases (RelA and SpoT) as well as its hydrolase (SpoT). A balanced ppGpp response is key for a proper adaptation during diauxic shift. The effects of one or the other ppGpp synthetase on diauxic shifts are abolished by addition of amino acids or succinate, although by different mechanisms. While amino acids control the RelA response, succinate blocks the uptake of the excreted acetate via SatP. Acetate is converted to Acetyl-CoA through the ackA-pta pathway, producing Ac-P as intermediate. Evidence of control of the ackA-pta operon as well as a correlation between ppGpp and Ac-P is shown. Finally, acetylation of proteins is shown to occur during a diauxic glucose–lactose shift.

The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates

“…Large array of effects of (p)ppGpp on diverse cellular processes. Numbers correspond to the references shown in the text (9,11,12,16,17,25,34,36,(43)(44)(45)(46)(48)(49)(50)(51)(52)(53)(54). Arrows represent positively regulated pathways, while crossed lines are negatively regulated pathways.…”

“…Both the synthesis and uptake of precursors are regulated by (p)ppGpp (9,11,17,48), resulting in the control of DNA, RNA, and protein. Moreover, central metabolism is also controlled by (p)ppGpp (49)(50)(51)(52). Mutations in central metabolism genes such as tktB, pgi, and pta change the thermosensitivities of dnaB8, dnaE486, and dnaA46 mutant alleles, respectively (53), but these mutations do not seem to affect DNA replication initiation (Fig.…”

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

“…Hence, zones with different acetate concentrations are formed, which is undesirable as acetate concentrations >2 g/L are already toxic for cells . Furthermore, acetate has several negative effects on growth and recombinant protein production as well as induces the cellular stress response, not the least because E. coli adjusts its expression pattern to growth on a secondary carbon source and vice versa . Hence, potentially less productive or less robust subpopulations can occur that reduce overall process efficiency.…”

Fatty acid starvation activates RelA by depleting lysine precursor pyruvate

“…After two freeze-thaw cycles, samples were centrifuged at 4 o C to pellet the cell debris, and 5 µl aliquots of the supernatants were applied to polyethyleneimine (PEI) cellulose thin layer chromatography plates (Merck, Sigma), resolved with 1.5 M KH2PO4 (pH 3.4) air dried and exposed by phosphorimaging (Amersham Typhoon phosphorimager). Quantification was done using ImageJ and relative aboundance of ppGpp and pppGpp was estimated as a percentage value relative to the total (pppGpp+ppGpp+GTP) amount as described (Fernandez-Coll & Cashel, 2018, Mechold et al, 2013.…”